| Assay Method Information | |
| | Kat6b Activity Assay |
| Description: | Kat6b inhibitory activities of the compounds described in the present invention were quantified using a Fluorescence Resonance Energy Transfer (TR-FRET) assay which measures acetylation of a synthetic, biotinylated Histone-H4-derived peptide by the enzyme.Recombinant human GST-tagged Kat6b protein (431-end, N-terminal GST-tag), purified from Baculovirus- infected insect cells (Sf9), was purchased from SignalChem (#K315-381 BG). Histone H4 peptide (amino acids 1-24, SGRGKGGKGLGKGGAKRHRK-VLRD-K(Btn)-amide), which was used as substrate, was synthesized by Biosyntan GmbH, Berlin, Germany. Acetyl Coenzym A was purchased from Sigma-Aldrich (#A-2056).Kat6b was incubated for 30 mins at 22°C in the presence of different concentrations of test substances (0 pM, and within the range 0.01 - 20 pM) in assay buffer [25 mM Tris/HCI pH 8, 1 mM EGTA, 2.5 mM Glutathion, 0.02% Chicken Albumin, 0.05% Pluronic F127, 25 mM NaCI, 500 nM H4 peptide and 600 nM Acetyl Coenzyme A],The reaction was stopped by addition of Detection Solution (25mM HEPES pH 7.5, 0.1 % BSA, 22nM SAXL665 (Cisbio #610SAXLE), 100pM Anacardic Acid (Enzo #ALX-270-381), 1 nM Anti-Histone H4 (ACETYL K8) Antibody (ABCAM #AB15823), 0.5 nM AntiRabbit IgG Eu (Perkin Elmer #AD0083).The fluorescence emission at 620 nm and 665 nm after excitation at 330-350 nm was measured in a TR-FRET measuring instrument, for instance a Rubystar or a Pherastar (both from BMG Lab Technologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emission at 665 nm and at 622 nm was used as indicator of the amount of acetylated peptide.The resulting data (ratio) were normalized, taking 0% inhibition as the mean value of control measurements where all reagents were included. In this case 50 nl DMSO were used instead of compounds. A 100% inhibition corresponded to the mean value of control measurements where all reagents except enzyme were included. IC50 values were determined by regression analysis based on a 4 parameter equation (minimum, maximum, ICM, Hill; Y = Max + (Min - Max) / (1 + (X/ICso) Hill) using the Screener Software (Genedata). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |